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Renal regulatory elements and methods of use thereof

a technology of renal regulatory elements and nucleic acids, applied in the field of nucleic acids, can solve the problems of inability or even death, damage to kidney tissue, impair kidney function, etc., and achieve the effect of treating or and preventing renal tissue injury

Inactive Publication Date: 2007-02-20
THE GENERAL HOSPITAL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019]The invention also includes a method for treating or preventing renal tissue injury. The method includes providing a cell that includes cis-acting KIM-1 regulatory sequence operably linked to a polypeptide coding sequence, and culturing the cell under conditions that allow for the expression of a therapeutic polypeptide-encoding sequence. The therapeutic polypeptide-encoding sequence is expressed, and the expressed polypeptide contacts the renal tissue, thereby treating or preventing renal tissue injury.

Problems solved by technology

Significant interruption in kidney function in an individual can lead to incapacitation or even death.
Disease or injury can impair kidney function.
In this type of injury, kidney tissue is damaged because of oxygen deprivation occurring as a result of interruption of blood flow to the kidneys.

Method used

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  • Renal regulatory elements and methods of use thereof
  • Renal regulatory elements and methods of use thereof
  • Renal regulatory elements and methods of use thereof

Examples

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Effect test

example 1

Cloning and Characterization of KIM-1 Derived Sequences

[0100]Cis-acting KIM-1 derived regulatory sequences were identified by screening a human genomic library with a 220 bp Not1-Kpn1 DNA fragment containing sequences in the 5′ region of the human KIM-1 cDNA. The human KIM-1 cDNA is described generally in PCT publication WO97 / 44460 and Ichimura et al., J. Biol. Chem. 273:4135–42, 1998.

[0101]The screening identified a genomic fragment of 8933 bp (SEQ ID NO:1). The sequence of the 8933 bp region is shown in FIGS. 1A–1H. A translational start codon is present beginning at nucleotide 8655.

[0102]A 4817 bp KpnI-KpnI fragment (SEQ ID NO:2), which corresponds to nucleotides 3796–8612 of FIGS. 1A–1H, was subcloned into a luciferase-encoding pGL3b expression vector (Promega Corp.) and named p4.8KIM / pGL3b. The p4.8KIM / pGL3K construct is shown schematically in FIG. 2B. The presence of the 4817 bp KpnI-KpnI fragment in the pLUC3 was found to increase levels of encoded luciferase in renal cells, ...

example 2

Expression in Kidney Cells of Reporter Sequences Operably Linked to KIM-1 Derived Sequences

[0105]Human genomic sequences from the 5′ region of the human KIM-1 gene were tested for their ability to direct expression of a reporter polypeptide in three kidney-derived cell lines COS cells, a cell line derived from African green monkey kidney fibroblasts; LC / PK cells, a cell line derived porcine kidney epithelial cell lines; and MDCK cells, a cell line derived from canine kidney epithelial cells.

[0106]The cell lines were transiently transfected with constructs containing various regions of DNA from the human KIM-1 gene linked to a reporter luciferase gene. These constructs were concomitantly transfected with pCMV driven β-galactosidase vectors to standardize transfection efficiency, and activity of luciferase and β-galactosidase was measured. Activities were calculated as luciferase / β-gal ratios. Relative activities were calculated as the ratio of construct activity to negative control, ...

example 3

Expression in Kidney Cells of Reporter Sequences Operably Linked to KIM-1 Derived Sequences Following Injury

[0124]Expression of luciferase encoded by 4.8 pKIM, 1.3 pKIM, and 0.5 pKIM was measured in transfected HK2 cells that had been subjected to chemical anoxia using cyanide and deoxyglucose. HK2 cells are derived from epithelial proximal tubule cells from human kidney.

[0125]A 12-well plate system (MULTIWELL™ 12-well, Becton-Dickson) was used in these studies. The culture medium used was EGM (Clontech). Prior to transformation, 2 ml medium per well was added to the cells, then removed by aspiration. HK2 cells were seeded at a density of 30,000 / well. Cells reached 80% confluence after 16–24 hours.

[0126]DNA was prepared by mixing 50 μl of serum free medium, 5 μg of DNA (2.5 μg luciferase construct, 0.5 μg β-galactosidase vector, 2 μg BlueScript vector), and 5 μl of SUPERFECT reagent (Qiagen Corp.), and incubating for 7 minutes. 300 μl of medium+10% FCS was added, and the mix was the...

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Abstract

Disclosed are cis-acting regulatory elements from a KIM-1 gene. The elements can be used to direct the expression of operably linked sequences in renal tissue.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a national phase filing under 35 U.S.C. § 371 of international application number PCT / US01 / 19295, filed Jun. 15, 2001, which claims the benefit of priority of provisional application No. 60 / 212,131, filed Jun. 16, 2000.STATEMENT OF GOVERNMENT INTEREST[0002]This invention was made with federal government support under grant #DK 39773. The United States government has certain rights in the invention.FIELD OF THE INVENTION[0003]The invention relates generally to nucleic acids and more particularly to nucleic acids which can be used to direct expression in renal tissue of operably linked sequences.BACKGROUND OF THE INVENTION[0004]Significant interruption in kidney function in an individual can lead to incapacitation or even death. Disease or injury can impair kidney function. An example of an injury that can damage kidneys is ischemic injury. In this type of injury, kidney tissue is damaged because of oxygen deprivation oc...

Claims

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Application Information

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IPC IPC(8): C07H21/04C12N15/00C12N15/63C12N15/85C12N15/09A61K35/76A61K38/00A61K48/00A61P9/12A61P13/12A61P17/00A61P37/02A61P39/00C07K14/705C12N1/19C12N5/10C12P21/02C12Q1/68
CPCC12N15/85C07K14/70503A01K2217/05A61K38/00A61K48/00C12N2830/008C07K2319/00C07K2319/02A61P13/12A61P17/00A61P37/02A61P39/00A61P9/12
Inventor SANICOLA-NADEL, MICHELEHESSION, CATHERINETIZARD, JR., RICHARDBONVENTRE, JOSEPH
Owner THE GENERAL HOSPITAL CORP
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